Vaccine adjuvants

ABSTRACT

The invention relates to a novel adjuvant  Mycobacterium  w and or its constituents and adjuvant containing composition, which contains antigen(s) with pharmaceutical acceptable carrier and its uses.  Mycobacterium  w and or its constituents when administered with antigen(s) to mammal results in enhanced immunogenicity of antigen. The enhanced immunogenicity manifests as humoral responses as well as cell mediated immunity. The adjuvant effect is seen with variety of antigens in various mammals irrespective of their immune status at the time of administration of  Mycobacterium  w and antigen containing composition. e.g. immune naïve or preimmunized status.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation patent application of U.S. Ser. No.10/583,731, filed on Feb. 19, 2008, now granted U.S. Pat. No. 8,048,434,which is U.S. National Stage Application of International ApplicationPCT/IB06/00978, filed Apr. 21, 2006, which claims the benefit ofpriority Indian Application No. 505/MUM/2005, filed Apr. 25, 2005, theentire contents of which are incorporated herein by reference.

FIELD OF INVENTION

The present invention pertains to novel adjuvants and compositionscontaining them with at least one antigen and methods for making andusing the same.

BACKGROUND OF TILE INVENTION

In case of infection specific immune response is concerned with therecognition and ultimate disposal of the antigen/immunogen in a highlydiscriminatory fashion. Specific immune responses are mediated throughtwo types of effectors mechanisms. One is mediated by antibody producedby lymphocytes (humoral response) and the other is mediated by speciallysensitized lymphocytes themselves (cell mediated immunity). The humoralresponses are mainly responsible for providing prophylaxis againstdisease (Prophylactic vaccine) while cell mediated immunity is mainlyresponsible for disease intervention (Therapeutic vaccine). Prophylacticvaccines are administered in anticipation of a disease. Therapeuticvaccines are administered in presence of an active disease.

The vaccine includes antigen (s) in a pharmaceutically acceptablecarrier.

An antigen is a substance that stimulates an immune response.

Varieties of antigens are described in textbooks, monographs andarticles. They include immunogens allergens, varieties of materialincluding or derived from pathogens and non pathogen's like virus,bacteria, fungi, parasites, material derived from tumors or cells. Thecells, organisms like virus, bacteria are also used in the intact forme.g. Polio, BCG etc. chemical composition of antigen is widely variableand include peptides of various kinds (like plain peptides,polypeptides, Lipopeptides etc.), polysaccharides, polysaccharideconjugates, lipids, glycolipids, carbohydrates, proteins, nucleic acidsor antigen encoding nucleic acids.

They are categorized in varieties of ways. Some of them are describedbelow.

Immunogen—Any substance that provokes the immune response whenintroduced into the body. An immunogen is always a macromolecule(protein, polysaccharide). Its ability to stimulate the immune reactiondepends on its commonness to the host, molecular size, chemicalcomposition and heterogeneity (e.g. similar to amino acids in aprotein).

Allergen—An allergen is a substance that causes the allergic reaction.It can be ingested, inhaled, injected or comes into contact with skin.

Antigens can be classified in order of their origins.

Exogenous antigens-Exogenous antigens are antigens that have entered thebody from the outside, for example by inhalation, ingestion, orinjection.

Endogenous antigens—Endogenous antigens are antigens that have beengenerated within the cell, as a result of normal cell metabolism, orbecause of viral or intracellular bacterial infection. The fragments arethen presented on the cell surface in the complex with class 1histocompatibility molecules.

Tumor antigens-Tumor antigens are those antigens that are presented bythe MHC I molecules on the surface of tumor cells. These antigens cansometimes be presented only by tumor cells and never by the normal ones.In this case, they arc called tumor-specific antigens and typicallyresult from a tumor specific mutation. More common are antigens that arepresented by tumor cells and normal cells, and they are calledtumor-associated antigens. Cytotoxic T lymphocytes that recognize theseantigens may be able to destroy the tumor cells before they proliferateor metastasize.

Tumor antigens can also be on the surface of the tumor in the form of,for example, a mutated receptor, in which case they will be recognizedby B cells.

Pathogen Associated Antigens

Antigens are derived from pathogens like virus, bacteria, fungus,parasites e.g. rabies, hepatitis B, mump, measles, tetanus, diphtheriaetc.

Antigens can be produced by recombinant technologies, extractionmethods, chemical synthesis, fermentation etc. it can be in the form ofa compound or an organism which is natural or genetically modified or afraction of an organism, which is naturally occurring or geneticallymodified. Nucleic acids are increasingly being developed and identifiedas antigens as in DNA vaccines. Antigens can be administered in the formof naked antigens, or encapsulated, coated form, conjugated, mixed,coupled and/or formulated with adjuvant.

Most of the vaccines when applied alone do not produce an adequateimmune stimulus, which is addressed by use of adjuvant e.g. alum inHepatitis B vaccine to provide desired effect.

-   -   Directly increasing the number of cells involved,    -   Assuring efficient processing of antigen, prolonging the        duration of antigen in immunized host,    -   Or by increasing the antibody synthesis by antibody synthesizing        cells.

Adjuvants are substances that enhance the immune response to antigens,but are not necessarily immunogenic themselves. Adjuvants may act byretaining the antigen locally near the site of administration to producea depot effect facilitating a slow, sustained release of antigen tocells of the immune system. Adjuvants can also attract cells of theimmune system to an antigen depot and stimulate such cells to elicitimmune responses.

A wide range of adjuvants provokes potent immune responses to antigens.These include saponins complexed to membrane protein antigens (immunestimulating complexes), pluronic polymers with mineral oil, killedMycobacteria in mineral oil, Freund's complete adjuvant, bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as lipid A, and liposomes. To efficiently induce humoral immuneresponse (HIR) and cell-mediated immunity (CMI), antigens are preferablyemulsified in adjuvants.

Currently the only adjuvant widely used in humans has been alum. Itcontains aluminum salts (alum) and has been useful for some vaccineslike hepatitis B, diphtheria, tetanus, toxoid etc., but not useful forothers like rabies MMR, typhoid etc. It fails to induce cell-mediatedimmunity. Aluminum hydroxide and aluminum phosphate is collectivelycommonly referred to as alum. Reports indicate that alum failed toimprove the effectiveness of whooping cough and typhoid vaccines andprovided only a slight effect with adenovirus vaccines. Problems withalum include induction of granulomas at the injection site andlot-to-lot variation of alum preparations (U.S. Pat. No. 6,861,410).

Other adjuvants, such as saponin, Qui A, and the water-in-oil adjuvant,Freund's with killed tubercle bacilli (Freund's complete) or withoutbacilli (Freund's incomplete), have had limited use in humans due totheir toxic effects; and, concerns have been raised as to undesirableeffects in animals. Most adjuvant formulations have been described butmost are never accepted for routine vaccines, mainly due to theirtoxicity and only few have been evaluated in humans

Complete Freund's adjuvant (CFA) is a powerful immunostimulatory agentthat has been successfully used with many antigens on an experimentalbasis. CFA includes three components: a mineral oil, an emulsifyingagent, and killed Mycobacterium tuberculosis. Aqueous antigen solutionsare mixed with these components to create a water-in-oil emulsion.Although effective as adjuvant, CFA causes severe side effects e.g.pain, abscess formation, fever etc. CFA, therefore, is not used inpreparation of commercial vaccines.

Incomplete Freund's adjuvant (IFA) is similar to CFA but does notinclude the bacterial component. It is an oil in water emulsion.However, evidence indicates that both the oil and emulsifier used in IFAcan cause tumors in mice.

Muramyl dipeptide (MDP) has been found to be the minimal unit of themycobacterial cell wall complex that generates the adjuvant activityobserved with CFA, e.g., Ellouz et al., Biochem. Biophys. Res. Commun.(1974) 59:1317. Several synthetic analogs of MDP have been generatedthat exhibit a wide range of adjuvant potency and side effects (Chedidet al., Prog. Allergy (1978) 25:63). Representative analogs of MDPinclude threonyl derivatives of MDP (Byars et al., Vaccine (1987)5:223), n-butyl derivatives of MDP (Chedid et al., Infect. Immun.35:417), and a lipophilic derivative of a muramyl tripeptide (Gisler etal., in Immunomodulations of Microbial Products and Related SyntheticCompounds (1981) Y. Yamamura and S. Kotani, eds., Excerpta Medica,Amsterdam, p. 167). One lipophilic derivative of MDP isN-acetylmuramyl-L-alanyl-D-isogluatrninyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE). The MTP-PE itself is able to act as an emulsifying agent togenerate stable oil-in-water emulsions. MTP-PE has been used in anemulsion of squalene with TWEEN 80, termed MTP-PE-LO (low oil), todeliver the herpes simplex virus gD antigen with effective results(Sanchez-Pescador et al., J. Immunol. (1988) 141:1720-1727), albeit withpoor physical stability.

Synthetic polymers are evaluated as adjuvants. These include the homo-and copolymers of lactic and glycolic acid, which have been used toproduce micro-spheres that encapsulate antigens (see Eldridge et al.,Mol. Immunol. 28:287-294 (1993)).

Nonionic block copolymers are another synthetic adjuvant beingevaluated. Adjuvant effects are investigated for low molecular weightcopolymers in oil-based emulsions and for high molecular weightcopolymers in aqueous formulations (Todd et al., Vaccine 15:564-570(1997)).

In fact, the adjuvant effect of most experimental adjuvants has beenassociated with the adverse effects they elicit. Adjuvants that act asimmunostimulators such as muramyl dipeptide, lipopolysaccaride, lipid A,monophosphoryl lipid A, and cytokines such as IL-2 and IL-12 can alsocause systemic side-effects (general toxicity, pyrogenicity), limitingtheir use.

The adjuvants using whole cells like insect cells (S. frugiperda) areknown (e.g., U.S. Pat. No. 6,224,882). The insects or the insect cellsinfected with some of the insect viruses/infectious agent or any othertype of infection, also it is not yet possible to identify whichinsect/insect cell is infected and which not hence the use of these(i.e., insect/insect cell) can result in low production and a possiblethreat of transmission of disease to human (WHO report January 2005).

In an article published in Vaccine (1999) 17; 2446-2452, Bacillus ofCalmette-Guerin (BCG) is used as adjuvant to rabies vaccination in mice.The experimental results show no improvement in serum neutralizingantibody titers in-group of mice immunized with BCG as adjuvant comparedto plain vaccine.

U.S. Pat. No. 6,355,414 describes acemannan polysaccharide as adjuvant.U.S. Pat. No. 6,306,404 describes adjuvant & vaccine compositions ofmono phosphoryl lipid A, sugar and optionally an amine-based surfactant.U.S. Pat. No. 6,231,859 describes saponin combination as adjuvant.Saponin adjuvants have high systemic toxicities, like haemolysis. TheU.S. Pat. No. 6,060,068 describes interleukin-2 as adjuvant to vaccines.U.S. Pat. No. 6,355,256 describes QS-21 & IL-12 as adjuvants.

U.S. Pat. Nos. 6,103,697, 6,228,373 & 6,228,374 describes peptides asadjuvants. JP 11106351, JP 9268130 & AU 780054 describe oil adjuvants.But in all these adjuvants are not demonstrated with wide variety ofantigens and mammals. Also the safety of these adjuvants is still to beconfirmed.

Side effects of currently used adjuvants includes: (1) sensitization totuberculin or any other antigen used in screening tests for infections;(2) presence in food animals of materials that cannot safely be ingestedby humans; (3) inflammatory, granulomatous, necrotizing, or otherunacceptable reactions at injection sites most notably as occurs withFreund's complete adjuvant; (4) pyrogenicity; (5) central nervous systemeffects and untoward behavioral effects; (6) impairment of growth; (7)arthritis; (8) increased vascular permeability and inflammatoryreactions in the eye; (9) induction of undesired autoimmune responsesand (10) immune suppression for adjuvant epitopes.

It is a long standing need of the industry to provide adjuvants that arefree of above-mentioned side effects. Surprisingly, it is observed thatMycobacterium w and/or its constituents fulfill the requirement ofadjuvant. Unlike Freund's adjuvant it provides immune stimulation inabsence of emulsion. It is also not associated with systemic sideeffects like fever, body ache, muscle pain etc.

Mycobacterium w is a rapidly growing Mycobacterium. Mycobacterium w is anon-pathogenic, cultivable, atypical Mycobacterium, with biochemicalproperties and fast growth characteristics resembling those belonging toRunyons group IV class of Mycobacteria in its metabolic and growthproperties but is not identical to those strains currently listed inthis group. It is therefore thought that (Mw) is an entirely new strain.The species identity of Mw has been defined by polymerase chain reactionDNA sequence determination.

The Mycobacterium w has been found useful for treatment of Leprosy,Tuberculosis (Publication No: WO03075825-2003-09-18), and also forcancer treatment (Publication No: WO03049667-2003-06-19).

There is currently a need to have better adjuvants. The better adjuvantsare needed in for improving efficacy of current vaccines like rabieswherein adjuvants like alum can not be used. They are needed to improvethe efficacy of adjuvant containing current vaccines e.g. Hepatitis Bvaccine containing alum. Better adjuvants are also needed to improveefficacy of various candidate vaccine so that they become effective andcan be effective used e.g. CEA containing vaccines. The new adjuvantsare also needed to provide novel vaccines for various new indicationslike vaccine for hepatic viral disease.

REFERENCES

-   1. Essential Immunology, Eight Edition-   Ivan Roitt, Black well Scientific publication.-   2. Vaccines, Third edition.-   S. Plotkein W. Orenstein, W.B. Saunder's company-   3. Vaccines Prospects & perspectives-   Harminder sigh, rajesh Bhatia, forward publishing company, Delhi-   4. Immunotherapy of cancer-   Mary L. Disis, Humana press, Totowa, N.J., USA.-   5. DNA vaccine-   Douglas B. Lowrie, Robert a Whalen, Humana press, Totowa N.J., USA.-   6. Handbook of cancer vaccines-   Micheal A. Morse, Timothy M. Clay, H. Kiva Lyerly. Humana press    Totowa N.J., USA.-   7. Cellular Microbiology-   Bian Henderson, Micheal Wilson, John wiley & sons.

SUMMARY OF INVENTION

Thus in accordance with the invention Mw preparations comprising ofwhole cell, and/or fraction there off individually or in combination,stimulates immune response against the formulated, mixed, or conjugatedimmunogen, for providing prophylactic and/or therapeutic vaccine.

The main object of invention is to provide adjuvant and adjuvantcontaining compositions that can stimulate the mammalian immune systemagainst a wide variety of antigen (s). It is another object of inventionto provide Mycobacterium w or its constituents as an adjuvant.

It is yet another objective to provide compositions containingMycobacterium w or its constituents as an adjuvants and antigen (s) in apharmaceutically acceptable carrier.

It is yet another objective to provide heat killed Mycobacterium was anadjuvant.

It is yet another objective to provide adjuvant and adjuvant tocontaining compositions made of whole Mycobacterium w and or itsconstituents.

It is yet another objective of present invention to provide a method forinducing or enhancing immunogenicity of an antigen in a mammal. Themethod includes administering to the mammal a vaccine composition thatincludes the antigen and a vaccine adjuvant composition that includes aneffective immunopotentiatory amount of Mw and/or its constituents.

It is yet another objective to provide adjuvant that stimulates immunesystem which when mixed formulated, conjugated, primed or any other typeof formulation with specific antigen/immunogen.

It is yet another objective to provide adjuvant composition whereinantigen (s) is selected from peptides, polypeptides, cells, cellextracts, polysaccharides, polysaccharide conjugates, lipids,glycolipids, carbohydrates, proteins, viruses, viral extracts, andantigen encoding nucleic acids.

It is yet another objective to provide adjuvant and adjuvant containingcompositions which comprises of Mycobacterium.

It is yet another objective to provide adjuvant and adjuvant containingcompositions that stimulates the cell mediated immune response.

It is yet another objective to provide adjuvant that stimulates theimmune system faster, better and for a longer period.

DETAILED DESCRIPTION

List of Figures and their Description

FIG. 1. Effect of immunization with Mw containing rabies vaccine inhorses with initial high titers.

FIG. 2. Effect of immunization with Mw containing rabies vaccine inhorses with initial low titers.

FIG. 3. Effect of immunization with Mw containing rabies vaccine onneutralizing antibody titers in pre-immunized horses before and afterthe treatment.

FIG. 4. Effect of multiple immunizations with rabies vaccine compared tosingle dose of Mw containing rabies vaccine on Anti rabies-Antibodytiters in Horse

FIG. 5. Effect of rabies antigen coated Mw vaccine on antibody responseagainst rabies virus in horse.

FIG. 6 Effect of antigen coated Mw vaccine on neutralizing antibodyresponse against rabies virus in horse.

FIG. 7 Effect of immunization with Rabies vaccines compared to Mwadjuvanted vaccine in mice.

FIG. 8. Anti-Rabies antibody titers by mouse neutralization test in miceimmunized with rabies vaccine and rabies vaccine formulated with Mw.

FIG. 9. Antibody response in healthy human volunteers against HBs Agimmunized intradermally, with Hepatitis B vaccine and Mw adjuvantedvaccine.

FIG. 10. Effect of 2^(nd) dose of immunization on Antibody responsefollowing hepatitis B vaccine and Mw adjuvanted vaccine.

FIG. 11. Booster effect of Mw-HBs Ag vaccine in human volunteers.

FIG. 12 Antibody responses in healthy human volunteers immunizedintramuscular with hepatitis B vaccine and Mw adjuvant.

FOLLOWING EXAMPLE DESCRIBED THE PROCESS OF OBTAININGCONSTITUENTS/FRACTIONS OF MYCOBACTERIUM W

Method of Growing Mycobacterium w

1. Culturing of Mycobacterium w

a) Preparation of Culture Medium

-   -   Mycobacterium w is cultured on solid medium more particularly on        LJ medium or more particularly in liquid medium like Middlebrook        medium or sautn's Liquid Medium.    -   Middlebrook medium is enriched for better yield. It can be        preferably enriched by addition of glucose, bactopeptone, and        bovine serum albumin and additives there on. They are used in        several ratios, preferably used in ratio of 20:30:2.    -   The enrichment medium is added to the Middlebrook medium in        several different ratio from 15:1 to 25:1 more preferably in the        ratio 20:1.

b) Bioreactor Preparation.

a. Preparation of Vessel

-   -   The inner contact parts of the vessel (Joints, Mechanical seals,        o-Ring/gasket, Grooves etc.) are properly cleaned to avoid any        contamination. The vessel is filled up with 0.1N NaOH and left        as such for 24 hrs to remove pyrogenic materials and other        contaminants. The vessel is then cleaned first with acidified        water, then three times with distilled water before preparing        medium.

b. Sterilization of Bioreactor

-   -   The bioreactor containing nine liters distilled water is        sterilized with live steam (Indirect/direct). Similarly the        bioreactor is sterilized once more with Middlebrook medium. The        other addition bottles, inlet/outlet air filters etc. are        autoclaved twice at 131.6° C. for 15 minutes. Before use these        are dried at 50° C. in an oven.

c. Environmental Parameters

-   -   i. Temperature: 37±0.5° C.    -   ii. pH: 6.7 to 6.8 initially

2. Harvesting and Concentrating

It is typically done at the end of 6^(th) day after culturing underaseptic condition but can be harvested at any time between 6 hrs to 15days. The concentration of cells (pelletization) is done bycentrifugation.

3. Washing of Cells

The pellet so obtained is washed for minimum three times with normalsaline. It can be washed with or without detergent containing fluid,which is preferably isotonic.

4. Adding pharmaceutically acceptable carrier.

Pyrogen free normal saline is added to pallet. Any other pyrogen freefluid can be used as pharmaceutical carrier. The carrier is added inamount so as to get desired concentration of active material in finalform.

5. Adding Preservative

To keep the product free from other contaminating bacteria for its selflife, preservative is added. Preferred preservative is thiomersal infinal concentration of 0.005% v/v to 0.1% w/v more preferably 0.01% w/v.

6. Terminal Sterilization

Terminal sterilization can be done by various physical methods likeapplication of heat or ionizing radiation or sterile filtration.

Heat can be in the form of dry heat or moist heat. It can also be in theform of boiling or pasteurization. Ionizing radiation can be ultravioletor gamma rays or microwave or any other form of ionizing radiation.

It is preferable to autoclave the final product. This can be done beforeor after final packaging.

7. Quality Control

-   -   a) The material is evaluated for purity and sterility.    -   b) The organisms are checked for acid fastness and gram        staining.    -   c) Inactivation test: This is done by culturing the product on        LJ medium to find out any living organism.    -   d) Pathogenicity and/or contamination with pathogen        -   The cultured organisms are infected to Balb/c mice. None of            the mice should die and all should remain healthy and gain            weight. There should not be any macroscopic or microscopic            lesions seen in liver, lung, spleen, or any other organs            when animals are sacrificed up to eight weeks following            treatment.    -   e) Biochemical Test    -   f) The organism is subjected to following tests    -   i. Urease    -   ii. Tween 80 hydrolysis    -   iii. Niacin Test    -   iv. Nitrate reduction test

The organism gives negative results in urease, tween 80 hydrolysis andniacin test. It is positive by nitrate reduction test

Following Examples Illustrates the Processes Used for ObtainingConstituents of Mycobacterium w the Scope of Invention is not Limited bythem

1. Cell Disruption

-   -   The cell disruption can be done by way of sonication or use of        high pressure fractionometer or by application of osmotic        pressure gradient. The disrupted cells were washed with        physiological saline and repelleted by centrifugation.

1. Solvent Extraction

-   -   Any organic solvent like alcohols, halogenated hydrocarbons,        acetone, phenol, isopropyl alcohol, acetic acid, hexane and/or        aromatic compound individually or in any combination thereof can        do the solvent extraction.

2. Enzymatic extraction.

-   -   The enzymatic extraction can he done by enzymes, which can        digest cell wall/membranes. They are typically proteolytic and        lytic in nature. Enzyme lysozymes, liticase and pronase are the        preferred enzymes.    -   The cell constituents/fractions of Mycobacterium w were used        alone in place of Mycobacterium w organisms and/or they were        added to the product containing Mycobacterium w. Addition of        cell constituents results in improved efficacy of the product.

Methods to Illustrate the Manufacture of Composition ContainingMycobacterium w and or its Constituents as an Adjuvant

The Mw as whole cell or fraction were formulated by mixing of Mw in anisotonic solution, fraction of Mw formulated in appropriate bufferand/or conjugated with an antigen or immunogen chemical coupling agentslike aldehydes, carbodiamides, anhydrides and any such compound.

Following examples demonstrate the invention and are not limiting forpurpose of invention.

EXAMPLE 1

Mycobacterium w cells were grown as mentioned above in Middlebrook mediaand were killed by autoclaving at 121.6° C. under steam pressure of 15Psi. The cells were suspended in sterile pyrogen free normal saline. Thecells were checked for sterility and purity. The cells were diluted to afinal concentration of 10⁹ cells per mL. The cells were mixed with theantigen, in one of the example the antigen is HBs ag. These mixtures areused as vaccine where Mw acts as adjuvant.

EXAMPLE 2

The heat killed Mw cells and/or its constituents were activated by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma) andwere coated with antigen, by mixing the activated cells with antigen.The molar ratio of antigen to cell used was in the range of 1:2 to 1:100preferably in the ratio of 1:50. This composition was used forimmunization where Mw and/or its constituents act as adjuvant.

Following Examples Illustrate the Adjuvant Effect

Following example describes the Method of using Mycobacterium w and itsconstituents/fractions as adjuvant. Following experiments demonstratethe adjuvant effect of present invention and they do not limit scope ofinvention.

-   -   A. Adjuvant effect as per present invention in preimmunized        horses receiving rabies vaccine for purpose of evaluating effect        compositions containing Mycobacterium w and/or its constituents        were formulated and evaluated for effect on antibody titer. All        single injections of control vaccine (Rabipur) as well as test        vaccine contained inactivated rabies virus more than or equal to        2.5 IU/ml. They were given in a different dosage (no. of        injections) or interval as mentioned. Antibody titers were        measured by ELISA method and were further evaluated with mice        neutralizing antibody test.

EXAMPLE 1

Immunization of Horse Against Rabies Using Mw Adjuvant:

Horses immunized with rabies vaccine (Rabipur) were re-immunized byadministering rabies vaccine (Rabipur) reconstituted with water or withMycobacterium w containing normal saline. Each horse received 1.0 ml ofreconstituted Rabipur intramuscularly in two doses of 0.5 ml each overeach shoulder. The blood was withdrawn at the beginning (zero day) andat the intervals of seven and fourteen days following re-immunization.The blood was analyzed for rabies antibody titer using ELISA againstreference standard. The blood samples were also evaluated forneutralizing rabies antibodies using mouse neutralizing antibody test.

FIG. 1 shows the effect of both vaccines on serum antibody levels inhorses, three in each group, having very low initial antibody titer. Thevaccine alone does not raise the antibody titer while vaccine with Mw iscapable of raising the titer to significant higher level on day 7. Thisincreases further on day 14.

FIG. 2 Shows the effect of both vaccines on serum antibody levels inhorses, two in each group, having high initial antibody titer. Thevaccine alone does not raise the antibody titer while vaccine with Mw iscapable of raising the titer to significant higher level on day 7. Thisincreases further on day 14.

When neutralizing antibodies were evaluated using mouse-neutralizingtest, it was observed that there was no effect on neutralizing antibodylevels in control group (receiving rabies vaccine alone) but there wassignificant improvement in a group receiving the vaccine withMycobacterium w. (test group) There was more than two fold rise inneutralizing antibody titer on day 14 compared to base level in testgroup (FIG. 3).

EXAMPLE 2

The test groups received rabies vaccine reconstituted with Mw while thecontrol group received only rabies vaccine. The test group was immunizedon day 0 with a single injection of vaccine as described earlier. Thecontrol group received four injections each of rabies vaccine on day 0,7 and 14. (Total of 12)

The results as shown in FIG. 4, the test group shows better immuneresponse compared to the control. The antibody response in test group isachieved with lesser amount of antigen compared to the control group.

EXAMPLE 3

Test vaccine as per invention was evaluated in same horses who receivedfour injection of standard rabies vaccine a times at the interval of 4weeks (16 injections total) prior to it. All received only one injectionof test vaccine which contained same load of antigen as contained insingle injection of rabies vaccine.

The antibody titer generated in horses with test vaccine is higher thanor equal to whatever is produced in same horse with four injections ofcontrol vaccine given every week for a total of 16 injections. Theantibody titer is also reached between 2 and 3 week in test groupcompared to five weeks in test group.

EXAMPLE 4

A group of five horses (Test Group) were immunized with rabies vaccineand the other group (Control Group) was immunized with the rabiesantigen coated Mw. Each horse received 1 ml of vaccine intramuscularly.The horses were bled at an interval of every 7 days for the period ofone month.

The titers of specific antibodies were measured by ELISA and MNT.

The titers of antibody in test group were significantly higher then inthe control group horses from day 7 onwards as shown in FIGS. 5 & 6.

EXAMPLE 5

Immunization of Mice Against Rabies

Mice were administered 0.2 ml of rabies vaccine (Rabipur) reconstitutedin water (control) or reconstituted with Mycobacterium w (Mw) containingnormal saline. In each group thirty mice were immunized. From each group5,10,15,20,25 and on 30th day five mice for each group were bled toobtain sera.

The vaccine was administered 0.2 ml intradermally divided into twoinjections of 0.1 ml each, over each side of back. The antibody titerwas evaluated subsequent to vaccine administration at the interval of 5days. The antibody titer was measured by ELISA (FIG. 7). The findingssuggest that addition of Mycobacterium w achieves higher antibody titterin comparison to control. It achieves such high titer very early. Thepeak value is achieved on day 10 in Mw group compared to day 15 incontrol group. On day 10 value achieved by Mw group is more than twicethat of control group.

In a control group after reaching the peak on day 15 it declines rapidlyand value achieved on day 20 is less than half of that achieved on day15 & is not detectable on day 25 & 30. In Mw group, value on day 20 ismore than twice compared to control group, which is maintained on day 25& day 30.

The samples were checked for specific isotypes of IgG.

These antibodies were having a major component of IgG2a isotypeantibody, as isotyped by IgG Isotyping kit (SIGMA).

EXAMPLE 6

Mouse Neutralizing Antibody Body Against Rabies.

Serum obtained on day 5, day 10, & day 25 from example five above wasused to detect mouse neutralizing antibodies by inoculating into brainof mice along with live rabies virus (Mouse neutralizing test).

On day 5, in both the groups the neutralizing antibody titer were nondetectable. On day 10 Mw group contained 1.35 IU/ml while control groupcontained 0.2975 of neutralizing antibodies. Even after the 25^(th) daythe specific neutralizing antibody titers were significantly also higherin-group with Mw as adjuvant then in control group. The titer of Mwgroup was 32 IU/mL while the control group just achieved the protectivetiter of 1.09 IU/mL.

This clearly shows the faster and specific induction of neutralizingantibody response to protective titers in 10 days in Mw group asindicated in the table 2 and FIG. 8.

TABLE 2 Effect of immunization with Immuvac Mw in mice Rabies controlvaccine Rabies vaccine Mw Day 5 Not detectable Not Detectable Day 100.2975 1.35 Day 25 1.09 32.00

B: Adjuvant Effect as Per Present Invention in Healthy Human VolunteersReceiving Hepatitis B Vaccine.

Compositions as per present inventions were prepared to have Hepatitis Bantigen (Test vaccine) and compared with Hepatitis-B vaccine(Engerix-B). Both (Test vaccine as well as control vaccine) contained 20microgram/ml of Hepatitis-B antigen. Antibody titers were measured usingcommercially available ELISA kit.

EXAMPLE 1 Effect of Single Intradermal Dose

After informed consent two groups of 15 human subjects male adults eachwere randomly selected and immunized by intradermal route. With a singleshot of either Hepatitis B vaccine (Engerix B) alone or as incorporatingMw the antigen of hepatitis B used was 2 mcg. Serum samples wereanalyzed for the antibody titer on week 1, 2, 3, and 4post-immunization. The results indicate that all of the volunteers incontrol group failed to rise against hepatitis B. The 10 IU/mL is theprotective level. On the other hand, group containing the vaccineincorporating Mw achieved significantly high titer (FIG. 9), more thanthe requisite levels of protective antibody titer.

EXAMPLE 2 Effect of Second Intradermal Dose

On 35^(th) day after first immunization, individuals with no responsereceived a second dose of immunization with test or control vaccineintradermally.

The antibody responses after second dose were determined at every sevendays. The anti-HBs antibodies in test group were significantly increasedcompared to the titers in control group as shown in FIG. 10. In controlgroup only one of six individuals receiving second dose of controlvaccine had rise in antibody titer.

EXAMPLE 3 Booster Effect of Single Dose of Intradermal Hepatitis BVaccine

Two individuals in with protective titers on day 0 received either atest vaccine or a control vaccine.

After immunization with respective vaccines, their antibody titers wereimproved in both.

EXAMPLE 4 Effect of Intramuscular Administration

In another example, 46 human male adult subjects, each were randomlyselected and immunized intramuscular with a single shot of eitherHepatitis B vaccine (control vaccine (Engerix 13)) alone or formulatedwith Mw (test vaccine). 15 volunteers received the control vaccine,while 31 volunteers were immunized with test vaccine. The antigen ofhepatitis B used was 20 mcG. Serum samples were analyzed for theantibody titer on week 1, 2, 3, 4, 6, and 7 post-immunization. Theresults indicate that all of the volunteers in control group failed torise against hepatitis B. The 10 IU/mL is the protective level. On theother hand, group containing the vaccine incorporating Mw achievedsignificantly high titer (FIG. 12). All achieved more than the requisitelevels of protective antibody titer with a single injection. Allreceived a second injection of control of test vaccine on day 28. Therewas a significant rise in antibody titer in test group (FIG. 12). Therise was also seen in control group but was not so remarkable.

EXAMPLE 5 Adjuvant Effect of Mw for Cell Mediated Immune ResponseAgainst HBs Ag in Mice

A group of five mice (test group) were immunized with Hepatitis Bvaccine (Engerix B) mixed with Mw and another group of five mice(control group) received appropriately diluted Hepatitis B vaccine. Theimmunization was performed sub-cutaneously.

Each mouse received 2 microgram of HBs antigen. The test group receivedthe same dose of 2-microgram antigen formulated in Mw cells.

After 15 days the mice were bled and PBMC were isolated. The isolatedcells were cultured in complete RPMI media with 10% FBS and 1 μG/mLconcanavalin A as mutagen at 37° C. and 7% CO₂ for 72 Hrs and stimulatedwith HBs antigen.

After 72 hrs the cells were harvested and are subjected to ELISPOT forIFN-γ, IL-2. The results were obtained with ELISPOT reader.

The results showed that the cells producing IFN-γ and IL-2 weresignificantly higher in test group compared to control group.

C: Adjuvant Effect as Per Present Invention to Demonstrate Effect onCell Mediated Immunity when Disease Antigens are Incorporated into theComposition.

EXAMPLE 1 Adjuvant Effect of Mw for Cell Mediated Immune ResponseAgainst Cancer Antigen Ca—19.9 in Mice

A group of five mice were immunized with CA—19.9 (Sigma) alone (controlgroup) or with a composition as per present invention. All mice wereimmunized with subcutaneous injections of 0.2 mL on lower back.

Each mouse received 10 IU of CA—19.9 antigen. The test group receivedthe same dose of 10 IU antigen formulated in Mw cells.

After 15 days the mice were bleed and PBMC were isolated. The isolatedcells were cultured in complete RPMI media with 10% FBS and 1 μG/mLconcanavalin A as mutagen at 37° C. and 7% CO₂ for 72 Hrs and stimulatedwith CA—19.9 antigen.

After 72 hrs the cells were harvested and are subjected to ELISPOT forIFN-γ and IL-2.

The results showed that number of cells secreting IFN-γ and IL-2 weresignificantly more in test group compared to control group.

EXAMPLE 2 Adjuvant Effect of Mw for Cell Mediated Immune ResponseAgainst Pneumococcal Antigen in Mice

A group of five mice were immunized with pneumococcal antigen alone(control group) or with a composition as per present invention. All micewere immunized with subcutaneous injections of 0.2 mL on lower backcontaining 0.1 mL of pneumococcal antigen.

After 15 days the mice were bled and PBMC were isolated. The isolatedcells were cultured in complete RPMI media with 10% FBS and 1 μG/mLconcanavalin A as mutagen at 37° C. and 7% CO₂ for 72 hrs and stimulatedwith pneumococcal antigen.

After 72 hrs the cells were harvested and are subjected to ELISPOT forIFN-γ, IL-2 and IL-12.

The results showed that number of cells producing IFN-γ were less in thecontrol group then the test group. The IL-2 response was observed onlyin test group.IL-12 secreting cells were significantly higher in testgroup.

EXAMPLE 3 Adjuvant Effect of Mw for Cell Mediated Immune ResponseAgainst Influenza Virus Antigen in Mice

A group of five mice were immunized with Influenza vaccine (Vaxigrip)mixed with Mw and another group of five mice received appropriatelydiluted influenza vaccine. All mice were immunized with subcutaneousinjections of 0.2 mL on lower back.

Each mouse received 0.10 ml of influenza vaccine. The test groupreceived the same dose of antigen formulated in Mw cells.

After 15 days the mice were bled and PBMC were isolated. The isolatedcells were stimulated in complete RPMI media with 10% FBS and 1 μG/mLconcanavalin A as mutagen at 37° C. and 7% CO₂ for 72 Hrs and stimulatedwith influenza vaccine.

After 72 hrs the cells were harvested and are subjected to ELISPOT forIFN-γ, IL-2 and IL-12.

The results showed that number of cells producing IFN-γ, IL-2 and IL-12in the test group were higher then the control group. The effect wasmaximum for IL-12 followed by IL-2 & IFN γ.

EXAMPLE 4 Adjuvant Effect of for Cell Mediated Immune Response AgainstSalmonella typhi Vi Antigen in Mice

A group of five mice (test group) were immunized with Salmonella typhiVi antigen mixed with Mw and the other group of five mice (controlgroup) received appropriately diluted Salmonella typhi Vi antigen. Allmice were immunized with subcutaneous injections of 0.2 mL on lowerback.

Each mouse received 0.1 ml Salmonella typhi Vi antigen. The test groupreceived the same dose of antigen formulated in Mw cells.

After 15 days the mice were bled and PBMC were isolated. The isolatedcells were cultured in complete RPMI media with 10% FBS and 1 μG/mLconcanavalin A as mutagen at 37° C. and 7% CO₂ for 72 hrs and stimulatedwith Salmonella typhi Vi antigen. After 72 hrs the cells were harvestedand are subjected to ELISPOT for IFN-γ, IL-2 and IL-12.

The results showed that in the test group had more number of cellsproducing IFN-γ, IL-2 and IL-12 then control group. The effect wasmaximum with 1-12 followed by IL-2 & IFN-γ.

EXAMPLE 5 Adjuvant Effect of Mw for Cell Mediated Immune ResponseAgainst Hepatitis A Antigen in Mice

A group of five mice (test group) were immunized with hepatitis Aantigen (Havrix) mixed with Mw and the other group of five mice (Controlgroup) received appropriately diluted hepatitis A antigen. All mice wereimmunized with subcutaneous injections of 0.2 ml on lower back.

Each mouse received 140 U of hepatitis A antigen. The test groupreceived the same dose of 140 U antigen formulated in Mw cells.

After 15 days the mice were bled and PBMC were isolated. The isolatedcells were cultured in complete RPMI media with 10% FBS and 1 μG/mLconcanavalin A as mutagen at 37° C. and 7% CO₂ for 72 hrs and stimulatedwith hepatitis A antigen.

After 72 hrs the cells were harvested and are subjected to ELISPOT forIFN-γ and IL-2. The results showed that in the test group number ofcells producing IL-2 and IFN-γ were significantly higher then thecontrol group.

The above examples demonstrate the working of present invention whenvariety of different kinds of antigens were incorporated. For some ofthem like rabies, typhoid, pneumococcal, currently there are noadjuvants incorporated into commercially available preparation due tothe fact that the current adjuvants do not provide desired effect. Italso demonstrate added adjuvant effect when combined with known adjuvantlike alum. Thus adjuvant of present invention appears to be universalwith better efficacy.

These examples clearly shows that the Mw when used as adjuvant canstimulate specific cell mediated immune response.

The results as shown in pre-immunized horses the single injections ofthe test vaccines elicits the antibody titer higher than the horsesimmunized with multiple injections of rabies vaccine.

The examples with human volunteers and horses also indicates that theprotective titers can be achieved by single immunization dose with testvaccine in 7 to 10 days while the similar titers can be achieved withconventional vaccine after one month or more with multiple injections.

The examples above show that the subject for the immune stimulation bypresent invention can be any mammal including small mice, large mammallike horse and human. The net response is identical in all the animals.The test compositions stimulates Cell Mediated Immunity is evident fromabove examples.

Mw when used along with rabies vaccine achieves a higher peak antibodylevel earlier, reducing time for first appearance of antibody, comparedto control group & maintains it for prolonged period.

Example above also shows the faster and specific induction ofneutralizing antibody response to protective titers in 10 days in Mwgroup as indicated in the table 2 and FIG. 7

None of the animals and human volunteers demonstrated any signs of localor generalized toxicity & vaccine was well tolerated. Identical resultsare also obtained when fractions/constituents of Mycobacterium w orwhole Mw is used.

Mw and/or its constituents there off can stimulate immune response inhuman against a specific antigen without any adverse effect. Also theroute of immunization does not matter to the immune response whenadministered with Mw as adjuvant.

This is indicative of the potential of Mw for eliciting enhanced andsustained humoral immune response for all antigens. As demonstrated byall the above examples the said adjuvant, Mw shall be used as whole orits constituents there off to enhance the stimulation of immune responseagainst practically any antigen/immunogen.

1. A method for generating an immune response against an antigen byadministration of a pharmaceutical composition comprising said antigenand Mycobacterium w and/or constituents thereof as an adjuvant.
 2. Themethod as claimed in claim 1 wherein the antigen(s) is a cell, a virus,a bacterium, a fungus, a parasite, a constituent thereof or acombination thereof.
 3. The method as claimed in claim 1 wherein theantigen is selected from the group consisting of one or more peptides,one or more polypeptides, one or more cells, one or more cell extracts,one or more polysaccharides, one or more polysaccharide conjugates, oneor more lipids, one or more glycolipids, one or more carbohydrates, oneor more proteins, one or more viruses, one or more viral extracts, andone or more nucleic acid-based antigen encoded in nucleic acids.
 4. Themethod as claimed in claim 1 wherein the antigen is an allergen.
 5. Themethod as claimed in claim 1 wherein the antigen is a tumor associatedantigen.
 6. The method as claimed in claim 1 wherein the antigen is atumor specific antigen.
 7. The method as claimed in claim 1, wherein theimmune response generated is a humoral immune response.
 8. The method asclaimed in claim 1, wherein the immune response generated is a cellmediated immune response.
 9. The method as claimed in claim 1 whereinthe administration of said Mycobacterium w and/or constituents thereofto a mammal induces or enhances immunogenicity of said antigen(s). 10.The method as claimed in claim 1 is used for the treatment of a disease.11. The method as claimed in claim 10 wherein the treatment comprisesgenerating immune response against a disease specific antigen.
 12. Themethod as claimed in claim 1 wherein the administration of saidMycobacterium w and/or constituents thereof induces or enhancesimmunogenicity of antigen(s) resulting in decreased morbidity &mortality associated with a disease.
 13. The method as claimed in claim1 wherein administration of said Mycobacterium w and/or constituentsthereof when combined with another therapy in a diseased mammal inducesor enhances immunogenicity of antigen(s) and results in decreasedmorbidity & mortality associated with disease.
 14. The method of claim 1wherein said pharmaceutical composition is administered by a parenteralroute.
 15. The method of claim 1 wherein the pharmaceutical compositionis administered by intramuscular, subcutaneous, or intradermal route.